A plasmin inhibitory peptide YHX-PIP-3 derived from whey protein and its application
By screening and synthesizing the β-lactoglobulin-derived plasmin inhibitory peptide YHX-PIP-3, the problem of inhibiting plasmin activity in milk was solved, the stability and shelf life of ultra-high temperature sterilized milk were achieved, and the development process was simplified.
Patent Information
- Application Number
- CN202311407194.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-10-27
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2043-10-27
AI Technical Summary
Existing technologies make it difficult to effectively inhibit the activity of plasmin in milk, resulting in gelation and shortened shelf life of milk after ultra-high temperature sterilization. In addition, the development of traditional plasmin inhibitors is highly complex.
An octa-peptide plasmin inhibitory peptide YHX-PIP-3 was screened from the β-lactoglobulin sequence through bioinformatics and molecular dynamics simulation. It was prepared and its inhibitory activity was verified by solid-phase synthesis and applied to ultra-high temperature sterilized dairy milk to inhibit plasmin activity.
It effectively inhibits the activity of plasmin, avoids milk gelation, extends the shelf life, simplifies the R&D process and reduces costs, and has good application prospects.
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Figure CN117447583B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of bioactive peptides, and in particular to a plasmin inhibitory peptide YHX-PIP-3 derived from whey protein and its application. Background Art
[0002] Plasmin is an endogenous enzyme secreted from plasma into milk through the mammary gland. It mainly exists in milk in the form of inactive zymogen. After heat treatment, the inactive zymogen can be activated into active plasmin. Plasmin is heat-stable and cannot be completely inactivated during ultra-high temperature sterilization.
[0003] Milk is generally sterilized at ultra-high temperatures during production. Active plasmin still exists in the milk after ultra-high temperature sterilization. During storage, plasmin will hydrolyze the casein in the milk, causing gelation, increasing the viscosity of the milk and reducing its fluidity, making the milk difficult to digest. In addition, plasmin is also the main cause of the bitter taste of cow's milk.
[0004] Research reports on controlling plasmin activity to date include the use of specific peptides and polyphenols as plasmin activity inhibitors; however, the development of specific plasmin inhibitors is challenging due to the complexity of the dynamic interactions and reactions in the plasmin system.
[0005] Therefore, how to solve the above technical problems and develop a new plasmin inhibitor with better plasmin inhibitory activity is a technical problem that technicians in this field urgently need to solve.
[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention
[0007] In response to the above technical problems, the embodiments of the present invention provide a plasmin inhibitory peptide YHX-PIP-3 derived from whey protein and its application to solve the problems raised in the above background technology.
[0008] The present invention provides the following technical solutions:
[0009] A plasmin inhibitory peptide YHX-PIP-3, the amino acid sequence of which is shown in SEQ ID NO.1.
[0010] Specifically, the plasmin-inhibiting peptide YHX-PIP-3 is a peptide segment with plasmin-inhibiting ability screened from the amino acid sequence of β-lactoglobulin. This peptide segment is an octapeptide sequence with the amino acid sequence of SEQ ID NO.1: TDYKKYLL, and is named plasmin-inhibiting peptide YHX-PIP-3.
[0011] Preferably, the plasmin inhibitory peptide YHX-PIP-3 is a water-soluble polypeptide; specifically, the plasmin inhibitory peptide YHX-PIP-3 is not only soluble in water, but also soluble in 0.01-0.1M PBS (pH 5.5-8) and DMSO.
[0012] Preferably, the plasmin inhibitory peptide YHX-PIP-3 has a molecular weight of 916.10 Da.
[0013] Preferably, the plasmin inhibitory peptide YHX-PIP-3 is prepared by solid phase synthesis.
[0014] A use of the plasmin inhibitory peptide YHX-PIP-3 as described above in the preparation of a plasmin inhibitor.
[0015] Preferably, the plasmin inhibitory peptide YHX-PIP-3 is used to prepare a plasmin inhibitor.
[0016] A use of the plasmin inhibitory peptide YHX-PIP-3 as described above in food additives.
[0017] Preferably, the plasmin inhibitory peptide YHX-PIP-3 is used as a milk additive. Adding the plasmin inhibitory peptide YHX-PIP-3 to ultra-high temperature sterilized milk can inhibit the activity of plasmin and avoid gelation of the milk system.
[0018] Specifically, ultra-high temperature sterilized milk refers to milk that has been sterilized at ultra-high temperature. The ultra-high temperature sterilization method refers to the instantaneous sterilization of milk by ultra-high temperature instantaneous sterilization (135℃ to 150℃, 4 to 15 seconds), which completely destroys the microorganisms and spores that can grow in it. It is a relatively common sterilization method at present.
[0019] Preferably, the plasmin inhibitory peptide YHX-PIP-3 is used to increase the shelf life of ultra-high temperature sterilized dairy milk.
[0020] A use of the plasmin inhibitory peptide YHX-PIP-3 as described above in the preparation of hemostatic drugs.
[0021] The present invention provides a plasmin inhibitory peptide YHX-PIP-3 derived from whey protein and its application, which has the following characteristics:
[0022] Beneficial effects:
[0023] 1. This study uses bioinformatics techniques, molecular docking, and molecular dynamics simulation to identify a plasmin-inhibiting peptide from the β-lactoglobulin sequence. This peptide is named plasmin-inhibiting peptide YHX-PIP-3.
[0024] 2. Studies have shown that the plasmin inhibitory peptide YHX-PIP-3 can inhibit the activity of plasmin. Adding it to ultra-high temperature milk can prevent the milk from precipitating and aging and gelling during storage, thereby enhancing the stability of the sterilized milk system and extending its shelf life.
[0025] 3. Compared with traditional peptide development methods, the plasmin-inhibiting peptide YHX-PIP-3 screening method used in this invention avoids the tedious steps of traditional peptide development, simplifies R&D costs, and the screened plasmin-inhibiting peptide YHX-PIP-3 still has strong plasmin inhibition ability in a real complex milk system.
[0026] 4. The plasmin inhibitory peptide YHX-PIP-3 screened by the present invention is derived from β-lactoglobulin and has good safety. This peptide segment contains only 8 amino acids, has a short synthetic sequence, is easy to synthesize, and has a clear mechanism of action. It can be synthesized quickly, in large quantities and at low cost, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the molecular docking and interaction interface between β-lactoglobulin and plasmin.
[0028] Figure 2 The Rg value changes of β-lactoglobulin and plasmin during molecular dynamics simulation of 200ns.
[0029] Figure 3 The inhibitory effect of peptide YHX-PIP-3 on plasmin.
[0030] Figure 4 The apparent changes of dUHT sterilized milk with or without YHX-PIP-3 after storage at 37°C for one week.
[0031] Figure 5 This is the effect of YHX-PIP-3 addition on the TSI value of dUHT milk stability. DETAILED DESCRIPTION
[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.
[0033] 1. Design principle of the present invention
[0034] 1. Research has found that β-lactoglobulin inhibits plasmin, likely due to its ability to act as a competitive substrate. Currently, specific peptide sequences within the β-lactoglobulin sequence have yet to be discovered. These peptides may act as important competitive inhibitors that bind to plasmin and reduce its activity.
[0035] With the development and application of bioinformatics technology, molecular docking and molecular dynamics simulation provide rich structural information of amino acid peptides of protein molecules, which provides technical support for the targeted screening of peptides with plasmin inhibitory ability from β-lactoglobulin sequences.
[0036] 2. In order to achieve the above object, the present invention adopts the following technical solutions:
[0037] (1) The structural data files of plasmin and β-lactoglobulin were obtained using the protein database;
[0038] (2) Molecular docking of β-lactoglobulin and plasmin was completed with the help of Z-DOCK (3.0.2) and Haddock (2.4), and the complex was subjected to 200 ns analytical dynamics simulation using Gromacs software;
[0039] (3) synthesizing a candidate plasmin inhibitory peptide by a solid-phase chemical synthesis method, and verifying the candidate plasmin inhibitory peptide by an inhibitory activity experiment to obtain the novel plasmin inhibitory peptide;
[0040] (4) The application of the selected plasmin inhibitory peptides in increasing the storage stability and extending the shelf life of directly ultra-high temperature sterilized milk (dUHT) was verified using a stability analyzer.
[0041] 2. The present invention will be further described in detail below with reference to specific embodiments.
[0042] Unless otherwise specified, the experimental methods and detection methods involved in the following embodiments are all conventional experimental methods and detection methods in the prior art.
[0043] Example 1 Molecular Docking and Molecular Dynamics Simulation of β-lactoglobulin and Plasmin
[0044] The structural files of plasmin and β-lactoglobulin (β-Lg) are from the AlphaFoldprotein protein structure database (E1B726) and the RCSBProteinDataBank protein database (5IO6). Before molecular docking, the structure of plasmin selected the amino acid 585-812 part in the plasminogen sequence structure, and used this part as a template for the interaction between plasmin and β-lactoglobulin. Plasmin and β-lactoglobulin were rigidly docked using Z-DOCK (3.0.2) software. PDBePISA (https: / / www.ebi.ac.uk / pdbe / pisa / ) was used to preliminarily analyze the binding energy and interacting amino acids of the docking results, and the docking model with the lowest Gibbs free energy was selected. It was then submitted to haddock for further flexible docking, and the model with the highest score in the docking results was selected. The results are as follows Figure 1 As shown. Figure 1 The β-lactoglobulin and plasmin complex in was used as the object of molecular dynamics simulation.
[0045] The molecular dynamics simulation is to make the β-lactoglobulin and plasmin complex in equilibrium, which is more in line with the actual state. The β-lactoglobulin and plasmin complex was simulated for 200ns at a temperature of 300K using Gromacs software to obtain the structure of the β-lactoglobulin and plasmin complex in equilibrium. Figure 2 As shown, after 200 ns of molecular dynamics simulation of the β-lactoglobulin and plasmin complex, the Rg of β-lactoglobulin fluctuated around 1.5 nm, while the Rg of plasmin fluctuated around 1.75 nm, indicating that the 200 ns molecular dynamics simulation made the complex obtained by the re-docking of β-lactoglobulin and plasmin in equilibrium.
[0046] Example 2 Screening of potential plasmin inhibitory peptides based on β-lactoglobulin sequence
[0047] The active site of plasmin is known to be a tripartite structure consisting of 624HIS, 667ASP, and 762SER. When the β-lactoglobulin-plasmin complex reaches equilibrium, the plasmin active site binds to the amino acid sequence of β-lactoglobulin. This binding site may competitively inhibit plasmin activity. Based on the amino acid sequence characteristics of plasmin inhibitors, which primarily contain lysine (Lys) and a small amount of arginine (Arg) at the P1 position, and in combination with the β-lactoglobulin amino acid sequence surrounding the plasmin active site, an octapeptide sequence, TDYKKYLL, was identified as a potential plasmin inhibitory peptide.
[0048] Example 3 Peptide Synthesis and Activity Verification
[0049] The screened peptide TDYKKYLL was synthesized using the peptide solid phase synthesis technology. After testing, the peptide had good water solubility. The effect of the peptide TDYKKYLL on the activity of plasmin was measured using the substrate colorimetric method. 0.01M phosphate buffer (pH 7.4) was prepared into a peptide solution with a concentration of 1 mg / mL. 50 μL of the peptide solution was mixed with 10 μL of 0.1U / mL plasmin, and then 140 μL of 0.1mM D-Val-Leu-Lys p-nitroaniline dihydrochloride was added. The absorbance at 405 nm was immediately measured using an enzyme reader. After shaking for three seconds, the absorbance was measured every 5 minutes, and the temperature was maintained at 37°C. In the control group sample, the peptide solution was replaced with phosphate buffer to measure the absorbance at 405 nm. As Figure 3 As shown, the addition of polypeptide YHX-PIP-3 can reduce the ability of plasmin to hydrolyze substrates, which indicates that the polypeptide TDYKKYLL extracted from β-lactoglobulin has the function of inhibiting the activity of plasmin.
[0050] Example 4 Application of plasmin inhibitory peptide in improving the stability of dUHT milk
[0051] In order to verify the inhibitory effect of peptide YHX-PIP-3 on plasmin in milk, the effect of peptide YHX-PIP-3 on the shelf life of milk was verified using dUHT sterilized milk. The heat treatment condition of fresh dUHT milk was 153℃ for 0.25s. Fresh dUHT milk and dUHT milk with YHX-PIP-3 were stored at 37℃ in the dark to simulate the accelerated storage process. The results are shown in Figure 2. Figure 4 As shown in the figure, after one week of storage, the appearance of the control milk and the milk with YHX-PIP-3 added was significantly different. The milk in the control milk underwent precipitation and stratification due to the hydrolysis of plasmin, while the milk system with YHX-PIP-3 added did not undergo significant changes compared to the storage on day 0.
[0052] The stability of the samples was further analyzed using a Formulaaction multiple light scattering instrument. Figure 5 As shown in the results, after one week of storage, the TSI index of the dUHT milk in the control group increased from 15 to 35, while the TSI index of the milk added with the peptide YHX-PIP-3 was around 5 and did not change significantly after 24 hours, indicating that the peptide of the present invention can effectively inhibit the activity of plasmin and extend the shelf life of dUHT milk.
[0053] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.
[0054] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.
Claims
1. A use of a plasmin inhibitory peptide YHX-PIP-3 in a food additive, characterized in that: The plasmin inhibitory peptide YHX-PIP-3 is used as a milk additive. Adding the plasmin inhibitory peptide YHX-PIP-3 to ultra-high temperature sterilized milk can inhibit the activity of plasmin and prevent the milk system from gelling. The amino acid sequence of the plasmin inhibitory peptide YHX-PIP-3 is shown in SEQ ID NO.
1.
2. The use of the plasmin inhibitory peptide YHX-PIP-3 in food additives according to claim 1, characterized in that: The plasmin inhibitory peptide YHX-PIP-3 is used for improving the shelf life of ultra-high temperature sterilized dairy milk.
3. Use of a plasmin inhibitory peptide YHX-PIP-3 in the preparation of a hemostatic drug; the amino acid sequence of the plasmin inhibitory peptide YHX-PIP-3 is shown in SEQ ID NO.1.